2,4-Dichlorophenoxyacetic acid (2,4-D) is one of the popular herbicides that is widely used in agriculture and can be found in food and water. A rapid and sensitive fluorescence polarization immunoassay (FPIA) was proposed for the detection of 2,4-D in juice and water. New tracers, 2,4-D-buthylenediamin fluoresceinthiocarbamyl (2,4-D-BDF) and 2,4-D-glycine aminofluorescein (2,4-D-GAF), were obtained and characterized. Monoclonal antibodies (MAb) obtained against 2,4-D were used as a recognition reagent. The kinetics of the interaction of MAb and tracers were studied, and the kinetic parameters of their binding were calculated. High specificity of binding of tracers and MAb was shown. In this work, an approach was elaborated on to reduce the detection limit of 2,4-D by the FPIA method by changing the volume of the studied sample. The optimized FPIA in a competitive format was characterized by the LODs of 2,4-D 8 and 0.4 ng/mL and the working ranges 30–3000 ng/mL and 3–300 ng/mL for juice and water, respectively. The entire test cycle (from sample receipt to evaluation of the analysis results) took only 20 min. The test for the recovery of 2,4-D in juice and water gave values from 95 to 120%, which demonstrated the reliability of the herbicide determination in real samples.

4-Chlorophenoxyacetic acid (4-CPA), a synthetic auxin analog, is employed in agriculture both as a plant growth regulator and as a constituent of herbicide formulations. Consequently, the establishment of simple and rapid detection methods is essential for effective environmental monitoring. This study reports the first development of a homogeneous fluorescence polarization immunoassay (FPIA) for the determination of 4-CPA. The monoclonal antibody (M1), raised against 4-CPA, was evaluated as a recognition element. Furthermore, two fluorescently labeled 4-CPA tracers—with ethylenediamine fluorescein thiocarbamate and aminohexylaminocarbonylfluorescein—were synthesized and purified, and their structures were unequivocally confirmed by high-performance liquid chromatography coupled with high-resolution mass spectrometric detection (HPLC-HRMS). Optimal concentrations of monoclonal antibodies and tracers were established, yielding a limit of detection of 1.2 ng/mL. The assay demonstrated a broad dynamic range of 2.3–300 ng/mL and a rapid analysis time of 15 min. Validation via the standard addition method in authentic open water samples resulted in recovery rates of 98–112%. To address the cross-reactivity with the prevalent herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), two novel strategies were devised and successfully implemented. The first approach involves the concurrent execution of two separate FPIAs—one for 2,4-D and one for 4-CPA—followed by the mathematical resolution of two analyte concentrations from the two measured binding values. The second strategy entails the preliminary selective removal of 2,4-D from sample matrices using affinity chromatography columns with immobilized anti-2,4-D antibodies prior to FPIA for 4-CPA. These proposed methodologies appear highly promising for overcoming the inherent limitations of traditional immunoassays when faced with significant cross-reactivity among structurally analogous compounds.